ES2649037T3 - Molecules with prolonged half-lives, compositions and uses thereof - Google Patents

Abstract

A modified molecule comprising a protein or non-protein agent and a constant IgG domain, in which the constant IgG domain comprises a human CH3 domain in which there is an amino acid substitution at amino acid residues 433, 434 and 436 , numbered according to the Kabat EU index, in which the modified molecule has a high half-life compared to the half-life of a molecule comprising the protein or non-protein agent and a corresponding IgG constant domain comprising a human CH3 domain not mutated, and in which the substitution in amino acid residue 433 is a substitution with a lysine, the substitution in amino acid residue 434 is a substitution with a phenylalanine and the substitution in amino acid residue 436 is a substitution with a histidine

Description

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the second sequence, then the molecules are identical in that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (ie,% identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are of the same length.

The determination of the percentage of identity between two sequences can also be carried out using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl Acad. Sci. U.S.A. 87: 2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl Acad Sci. U.S.A. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403. Nucleotide searches can be performed with BLAST with the set of NBLAST nucleotide program parameters, for example, for punctuation = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. Proteins can be searched for with BLAST with the parameter set of the XBLAST program, for example, for punctuation = 50, word length = 3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain alignments with gaps for comparison purposes, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). If the BLAST, Gapped BLAST and PSI-Blast programs are used, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used (see, for example, http: //www.ncbi.nlm.nih .gov). Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) that is part of the GCG sequence alignment software package. If the ALIGN program is used to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used.

The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percentage of identity, usually only exact correspondences are counted.

4. Description of the figures

FIG. 1 shows the structure of the hinge-Fc region of IgG indicating the locations of the residues identified by are involved in the interaction with the FcRn receptor (Ghetie et al., Immunology Today, 18 (12): 592-598, 1997) . FIG. 2 shows the amino acid sequence of the hinge-Fc region of human IgG1 (SEQ ID NO: 83) containing a hinge region (SEQ ID NO: 82), domain CH2 (SEQ ID NO: 80) and domain CH3 (SEQ ID NO: 81). FIG. 3 (A and B) show the amino acid sequences of (A) human FcRn (SEQ ID NO: 84) and (B) mouse FcRn (SEQ ID NO: 85), respectively. FIG. 4 shows the amino acid sequence of the hinge-Fc region of human IgG1 (SEQ ID NO: 83), in which non-mutated moieties that are mutated by amino acid substitutions are indicated in bold underlined. FIG. 5 shows a schematic diagram of the immunopurification process for the modified hinge-Fc library presented in phage. FIG. 6 shows a summary of the appearance of mutant moieties selected at the variant positions in the screened libraries. FIG. 7 (A-D). (A) shows the binding of murine FcRn to immobilized IgG1 having substitutions M252Y / S254T / T256E. Murine FcRn was injected at 10 different concentrations ranging from 1 nM to 556 nM on a surface on which 4000 resonance units (UR) of IgG1 had been coupled. After equilibrium is reached, residual bound protein was eluted with a PBS pulse, pH 7.4. (B) shows the binding of human FcRn to immobilized IgG1 / M252Y / S254T / T256E. Murine FcRn was injected at 8 different concentrations ranging from 71 nM to 2.86 µM on a surface on which 1000 UR of IgG1 had been coupled. After equilibrium is reached, residual bound protein was eluted with a PBS pulse, pH 7.4. (C) and (D) show Scatchard analysis of the data in (A) and (B), respectively, after correction for non-specific binding. Req is the corrected equilibrium response at a given concentration C. The representations are linear with correlation coefficients of 0.97 and 0.998, respectively. Apparent Kd are 24 nM and 225 nM, respectively. FIG. 8 (A-H). (A) - (D) show the results of BIAcore analysis of the murine FcRn binding at pH 6.0 and pH 7.4 to (A) non-mutated human IgG1, (B) M252Y / S254T / T256E, (C) H433K / N434F / Y436H, and (D) G385D / G386P / N389S, respectively, after correction for non-specific binding. Murine FcRn was injected at a concentration of 1.1 µm on a surface on which 1000 UR of non-mutated IgG1, 1000 UR of M252Y / S254T / T256E, 955 UR of H433K / N434F / Y436H and 939 UR of G385D had been coupled / Q386P / N389S. (E) - (H) show the results of BIAcore analysis of the binding of human FcRn at pH 6.0 and pH 7.4 to (E) non-mutated human IgG1, (F) M252Y / S254T / T256E, (G) H433K / N434F / Y436H, and (H) G385D / Q386P / N389S, respectively, after correction for non-specific binding. Human FcRn was injected at a concentration of 1.4 µm on a surface on which 1000 UR of non-mutated IgG1, 1000 UR of M252Y / S254T / T256E, 955 UR of H433K / N434F / Y436H and 939 UR of G385D had been coupled / Q386P / N389S.

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FIG. 9 shows the surface space filling model of the Fc fragment of a human IgG1 based on the human IgG1 structure of Deisenhofer, 1981, Biochemistry 20: 2361-2370. The residues are color-coded according to the free stabilization energy increase of the Fc-FcRn complex: red, it was found that substitutions at these positions increased affinity a factor of at least 2.5 times in the human Fc / FcRn interaction and at least 5 times in the mouse Fc / FcRn interaction; blue, substitutions in those positions were found to increase affinity a factor of less than 2 times in both the human Fc-FcRn and mouse Fc-FcRn interaction. The figure was drawn using Swiss-PdbViewer (Guex and Peitsch, 1997, Electrophoresis 18: 2714-2723). FIG. 10 shows changes in serum concentration ([Mab] ng / ml) over time (in days) of antibody that has a constant non-mutated domain (SYNAGIS®) (white squares), or constant domains with the following mutations: M252Y / S254T / T256E (white circles), G385D / Q386P / N389S (filled squares) and H433K / N434F / Y436H (filled circles). The antibody concentration was determined using anti-human IgG ELISA.

5. Detailed description of the invention

The present invention relates to modified molecules, particularly proteins, more particularly immunoglobulins, which have a high half-life in vivo and comprise a constant IgG domain, which bind to an FcRn (preferably an Fc or hinge-Fc domain), which contain one or more amino acid modifications with respect to a constant domain of non-mutated IgG, whose modifications increase the affinity of the constant domain of IgG, or fragment thereof, by FcRn. In a preferred embodiment, the invention particularly relates to the modification of human or humanized IgG and other bioactive molecules that contain human IgG FcRn binding portions, which have particular use in human therapy, prophylaxis and diagnosis.

5.1 Molecules with high half-lives in vivo

The present invention is based on the identification of amino acid modifications, in particular portions of the constant domain of IgG that interact with FcRn, whose modifications increase the affinity of IgG, or fragment thereof, for FcRn. Accordingly, the invention relates to modified molecules, preferably proteins, more preferably immunoglobulins, which comprise a constant domain of IgG, which have one or more amino acid substitutions, in one or more regions that interact with FcRn, whose modifications increase affinity. of the IgG or fragment thereof, by FcRn, and also increase the in vivo half-life of the molecule. In preferred embodiments, the one or more amino acid modifications are made in one or more of residues 251-256, 285-290, 308-314, 385-389 and 428-436 of the IgG hinge-Fc region (for example , as in the hinge-Fc region of human IgG1 represented in Figure 4, SEQ ID NO: 83), or analogous moieties thereof, as determined by alignment of amino acid sequences, in other hinge-Fc regions of IgG . In a preferred embodiment, amino acid modifications are made in a constant domain of human IgG, or FcRn binding domain thereof. In a certain embodiment, modifications are not made to residues 252, 254 or 256 (ie, all are made to one or more of residues 251, 253, 255, 285-290, 308-314, 385-389 or 428436) of the constant domain of IgG. In a more preferred embodiment, the amino acid modifications are not the substitution with leucine at residue 252, with serine at 254 and / or with phenylalanine at position 256. In particular, in preferred embodiments, such modifications are made when the constant domain IgG, hinge-Fc domain, hinge-Fc domain or other FcRn binding fragment thereof derives from a mouse.

The amino acid modifications are substitutions in one or more of residues 251-256, 285-290, 308-314, 385389 and 428-436, which increase the in vivo half-life of the constant domain of IgG, and any molecule bound thereto, and increase the affinity of the IgG, or fragment thereof, for FcRn. Preferably, the one or more modifications also produce a higher binding affinity of the constant domain for FcRn at pH 6.0 than at pH 7.4. In other embodiments, the modifications alter (i.e. increase or decrease) the bioavailability of the molecule, in particular, alter (i.e. increase or decrease) the transport (or concentration or half-life) of the molecule to mucosal surfaces ( for example, from the lungs) or other portions of a target tissue. In a preferred embodiment, amino acid modifications alter (preferably, increase) the transport or concentration or half-life of the molecule to the lungs. In other embodiments, amino acid modifications alter (preferably, increase) the transport (or concentration or half-life) of the molecule to the heart, pancreas, liver, kidney, bladder, stomach, large or small intestine, respiratory tract, lymph nodes, tissue nervous (central and / or peripheral nervous tissue), muscle, epidermis, bone, cartilage, joints, blood vessels, bone marrow, prostate, ovary, uterus, tumor or cancer tissue, etc. In a preferred embodiment, the amino acid modifications do not suppress, or, more preferably, do not alter, other immune effector or receptor binding functions of the constant domain, for example, but are not limited to complement fixation, ADCC and FcγRI binding. , FcγRII, and FcγRIII, as can be determined by methods well known and routine in the art. In another preferred embodiment, the modified FcRn binding fragment of the constant domain does not contain sequences that mediate immune effector functions or other receptor binding. Such fragments may be particularly useful for conjugation with a non-IgG or non-immunoglobulin molecule to increase the in vivo half-life thereof. In yet another embodiment, the effector functions are selectively altered (for example, to reduce or increase the effector functions).

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In preferred embodiments, amino acid modifications are substitutions at one or more of residues 308, 309, 311, 312 and 314, particularly a substitution with threonine at position 308, proline at position 309, serine at position 311, acidic aspartic at position 312 and / or leucine at position 314. Alternatively, the modification is the substitution with an isoleucine at position 308, proline at position 309 and / or a glutamic acid at position 311. In yet another embodiment, residues in one or more of positions 308, 309, 311, 312 and 314 are substituted with threonine, proline, leucine, alanine, and alanine, respectively. Accordingly, in certain embodiments the remainder in position 308 is substituted with threonine or isoleucine, the remainder in position 309 is substituted with proline, the remainder in position 311 is substituted with serine, glutamic acid or leucine, the remainder in the position 312 is substituted with alanine, and / or the rest in position 314 is substituted with leucine or alanine. In a preferred embodiment, the substitution is a threonine at position 308, a proline at position 309, a serine at position 311, an aspartic acid at position 312 and / or a leucine at position 314.

In preferred embodiments, amino acid modifications are substitutions at one or more of residues 251, 252, 254, 255 and 256. In specific embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with tyrosine, phenylalanine , serine, tryptophan or threonine, the remainder 254 is substituted with threonine or serine, the remainder 255 is substituted with arginine, leucine, glycine or isoleucine, and / or the remainder 256 is substituted with serine, arginine, glutamine, glutamic acid, acid aspartic, alanine, asparagine or threonine. In a more specific embodiment, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, residue 255 is substituted with arginine, and / or residue 256 is substituted with acid glutamic

In preferred embodiments, amino acid modifications are substitutions at one or more of residues 428, 433, 434 and 436. In specific embodiments, moiety 428 is substituted with threonine, methionine, leucine, phenylalanine or serine.

In preferred embodiments, amino acid modifications are substitutions at one or more of moieties 385, 386, 387 and 389, more specifically, which have substitutions at one or more of these positions. In specific embodiments, moiety 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine, moiety 386 is substituted with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine or methionine, the remainder 387 is substituted with arginine, proline, histidine, serine, threonine or alanine, and / or the remainder 389 is substituted with proline, serine or asparagine. In more specific embodiments, residues in one or more of positions 385, 386, 387 and 389 are substituted with arginine, threonine, arginine and proline, respectively. In yet another specific embodiment, residues in one or more of positions 385, 386 and 389 are substituted with aspartic acid, proline and serine, respectively.

In particular embodiments, amino acid modifications are made in one or a combination of residues 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434 and / or 436, particularly where the modifications are one or more of the amino acid substitutions described immediately above for these moieties. In a preferred embodiment, the molecule of the invention contains an Fc region, or FcRn binding domain thereof, which has one or more of the following substitutions: leucine at residue 251, tyrosine at residue 252, threonine or serine in the remainder 254, arginine in the remainder 255, threonine in the remainder 308, proline in the remainder 309, serine in the remainder 311, aspartic acid in the remainder 312, leucine in the remainder 314, arginine in the remainder 385, threonine in the rest remainder 386, arginine in remainder 387, proline in remainder 389, methionine in remainder 428.

In a preferred embodiment, the FcRn binding domain has a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or 18 of residues 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434 and / or

436

Amino acid modifications can be made by any method known in the art and many such methods are well known and routine to the skilled person. For example, but not by way of limitation, amino acid substitutions, deletions and insertions can be carried out using any well-known PCR-based technique. Amino acid substitutions can be made by site-directed mutagenesis (see, for example, Zoller and Smith, Nucl. Acids Res. 10: 6487-6500, 1982; Kunkel, Proc. Natl. Acad. Sci USA 82: 488, 1985). Mutants that produce high affinity for FcRn and high half-life can be easily screened in vivo using well-known and routine assays, such as those described in Section 5.11, below. In a preferred method, amino acid substitutions are introduced into one or more moieties in the constant IgG domain or FcRn binding fragment thereof and the mutated constant domains or fragments are expressed on the bacteriophage surface which are then screened for high affinity. binding by FcRn (see, in particular, Section 5.2 and 5.11, below).

Preferably, the amino acid residues to be modified are residues exposed on the surface. Additionally, in the preparation of amino acid substitutions, preferably the amino acid residue to be substituted is a conservative amino acid substitution, for example, a polar moiety is substituted with a polar moiety, a hydrophilic moiety with a hydrophilic moiety, the moiety hydrophobic with a hydrophobic moiety, a positively charged moiety with a positively charged moiety, or a negatively charged moiety with a negatively charged moiety. In addition, preferably, the remaining amino acid to be modified is not highly or completely conserved across species and / or is critical to maintain the tertiary structure of the domain.

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constant or for binding to FcRn. For example, but not by way of limitation, histidine modification in moiety 310 is not preferred.

Fc domain specific mutants that have high affinity for FcRn that were isolated after the third

5 round of immunopurification (as described in Section 6) from a library of mutant human IgG1 molecules that had mutations at residues 308-314 (histidine at position 310 and tryptophan at position 313 are fixed), those isolated after the fifth round of immunopurification of the library for residues 251-256 (isoleucine at position 253 is fixed), those isolated after the fourth round of immunopurification of the library for residues 428-436 (histidine at position 429, glutamic acid at position 430, alanine in

10 position 431, leucine at position 432 and histidine at position 435 are fixed), and those isolated after the sixth round of immunopurification of the library for residues 385-389 (glutamic acid at position 388 is fixed) listed in Table I. Non-mutant human IgG1 has a Val-Leu-His-Gln-Asp-Trp-Leu sequence (SEQ ID NO: 86) at positions 308-314, Leu-Met-Ile-Ser-Arg -Thr (SEQ ID NO: 87) at positions 251-256, Met-His-Glu-Ala-Leu-His-Asn-His-Tyr (SEQ ID NO: 88) at positions 428-436 and Gly-Gln -Pro-Glu-Asn

15 (SEQ ID NO: 89) in positions 385-389.

Table I

MUTANTS ISOLATED BY IMMUNOPURIFICATION

LIBRARY

MUTANTS *

251-256

Leu Tyr Ile Thr Arg Glu (SEQ ID NO: 90)

Leu Tyr Ile Ser Arg Thr (SEQ ID NO: 91)

Leu Tyr Ile Ser Arg Ser (SEQ ID NO: 92)

Leu Tyr Ile Ser Arg Arg (SEQ ID NO: 93)

Leu Tyr Ile Ser Arg Gln (SEQ ID NO: 94)

Leu Trp Ile Ser Arg Thr (SEQ ID NO: 95)

Leu Tyr Ile Ser Leu Gln (SEQ ID NO: 96)

Leu Phe Ile Ser Arg Asp (SEQ ID NO: 97)

Leu Phe Ile Ser Arg Thr (SEQ ID NO: 98)

Leu Phe Ile Ser Arg Arg (SEQ ID NO: 99)

Leu Phe Ile Thr Gly Ala (SEQ ID NO: 100)

Leu Ser Ile Ser Arg Glu (SEQ ID NO: 101)

Arg Thr Ile Ser Ile Ser (SEQ ID NO: 102)

308-314

Thr Pro His Ser Asp Trp Leu (SEQ ID NO: 103)

Ile Pro His Glu Asp Trp Leu (SEQ ID NO: 104)

385-389

Arg Thr Arg Glu Pro (SEQ ID NO: 105)

Asp Pro Pro Glu Ser (SEQ ID NO: 106)

Ser Asp Pro Glu Pro (SEQ ID NO: 107)

Thr Ser His Glu Asn (SEQ ID NO: 108)

Ser Lys Ser Glu Asn (SEQ ID NO: 109)

His Arg Ser Glu Asn (SEQ ID NO: 110)

Lys Ile Arg Glu Asn (SEQ ID NO: 111)

Gly Ile Thr Glu Ser (SEQ ID NO: 112)

Ser Met Ala Glu Pro (SEQ ID NO: 113)

428-436

Met His Glu Ala Leu Arg Tyr His His (SEQ ID NO: 114)

Met His Glu Ala Leu His Phe His His (SEQ ID NO: 115)

Met His Glu Ala Leu Lys Phe His His (SEQ ID NO: 116)

Met His Glu Ala Leu Ser Tyr His Arg (SEQ ID NO: 117)

Thr His Glu Ala Leu His Tyr His Thr (SEQ ID NO: 118)

Met His Glu Ala Leu His Tyr His Tyr (SEQ ID NO: 119)

* Substitution remains are indicated in bold

The sequences underlined in Table I correspond to the sequences that occurred 10 to 20 times in the

20 final round of immunopurification and italicized sequences correspond to the sequences that occurred 2 to 5 times in the final round of immunopurification. Those sequences that are neither underlined nor in italics occurred once in the final round of immunopurification.

In a preferred embodiment, the invention provides modified immunoglobulin molecules (e.g.,

25 various antibodies) that have high half-life in vivo and affinity for FcRn with respect to unmodified molecules (and, in preferred embodiments, altered bioavailability such as elevated or reduced transport to mucosal surfaces or other target tissues). Such immunoglobulin molecules include IgG molecules that naturally contain an FcRn binding domain and other non-IgG immunoglobulins (eg, IgE, IgM, IgD, IgA and IgY), or immunoglobulin fragments that have been manipulated to contain a fragment of union to

FcRn (i.e., fusion proteins comprising non-IgG immunoglobulin or a portion thereof and a

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binding domain to FcRn). In both cases, the FcRn binding domain has one or more amino acid modifications that increase the affinity of the constant domain fragment for FcRn.

Modified immunoglobulins include any immunoglobulin molecule that binds (preferably, immunospecifically, that is, competes out of non-specific binding), as determined by immunoassays well known in the art to assay for specific antigen-antibody binding) to an antigen. and contains an FcRn binding fragment. Such antibodies include, but are not limited to, polyclonal, monoclonal, bispecific, multispecific, human, humanized, chimeric, single chain antibodies, Fab fragments, F (ab ') 2 fragments, disulfide-linked Fvs and fragments containing either a VL or VH domain or even a complementarity determining region (CDR) that specifically binds to an antigen, in certain cases, manipulated to contain or fused with an FcRn binding domain.

The IgG molecules of the invention, and FcRn binding fragments thereof, are preferably IgG subclasses of IgG, but they can also be any other IgG subclass of given animals. For example, in humans, the IgG class includes IgG1, IgG2, IgG3 and IgG4; and mouse IgG includes IgG1, IgG2a, IgG2b, IgG2c and IgG3. It is known that certain subclasses of IgG, for example, mouse IgG2b and IgG2c, have higher removal rates than, for example, IgG1 (Medesan et al., Eur. J. Immunol., 28: 2092-2100, 1998) . Thus, if IgG subclasses other than IgG1 are used, it may be advantageous to replace one or more of the residues, particularly in the CH2 and CH3 domains, which differ from the IgG1 sequence with those of IgG1, thereby increasing the half-life in vivo. of the other types of IgG.

The immunoglobulins (and other proteins used herein) can be of any animal origin that includes birds and mammals. Preferably, the antibodies are human, rodent (eg, mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from transgenic animals for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described below and, for example, in US Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention can be monospecific, bispecific, triespecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide or may be specific for heterologous epitopes, such as a heterologous polypeptide or solid support material. See, for example, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol., 147: 60-69, 1991; U.S. patents No. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol., 148: 1547-1553, 1992.

Antibodies of the invention include derivatives that are otherwise modified, that is, by covalent binding of any type of molecule to the antibody such that covalent binding does not prevent the antibody from binding to the antigen and / or generating an anti-idiotypic response. . For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, ligand binding. cell or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more non-classical amino acids.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques that include those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981). The term "monoclonal antibody", as used herein, is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, which includes any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods of production and screening of specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell that expresses such an antigen. Once an immune response is detected, for example, antibodies specific for the antigen are detected in the mouse serum, the spleen is collected from the mouse and the splenocytes are isolated. Splenocytes are then fused by well known techniques with any suitable myeloma cell. The hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding to the antigen. Ascitic fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.

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Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, Fab and F (ab ') 2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') 2 fragments). F (ab ') 2 fragments contain the complete light chain and the variable region, the CH1 region and the hinge region of the heavy chain.

For example, antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are presented on the surface of phage particles that carry the polynucleotide sequences that encode them. In a particular embodiment, such a phage can be used to present antigen binding domains, such as Fab and Fv or Fv stabilized by disulfide bond, expressed from a combinatorial repertoire or library of antibodies (eg, human or murine). The phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified with antigen, for example, using labeled antigen or antigen bound or captured to a solid or pearl surface. The phage used in these methods is usually filamentous phage, which includes fd and M13. Antigen binding domains are expressed as a protein recombinantly fused to either phage gene III or gene VIII protein. Alternatively, the modified FcRn binding portion of immunoglobulins of the present invention can also be expressed in a phage display system.

Examples of phage display methods that can be used to prepare the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods, 182: 41-50, 1995; Ames et al., J. Immunol. Methods, 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol., 24: 952-958, 1994; Persic et al., Gene, 187: 9-18, 1997; Burton et al., Advances in Immunology, 57: 191280, 1994; PCT application No. PCT / GB91 / 01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401; and U.S. patents No. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;

5,733,743 and 5,969,108;

As described in the references above, after phage selection, phage antibody coding regions can be isolated and used to generate complete antibodies, including human antibodies, or any other desired fragment, and expressed in any desired host, which It includes mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, techniques can also be employed to recombinantly produce Fab, Fab 'and F (ab') 2 fragments using methods known in the art such as those disclosed in PCT Publication WO 92/22324; Mullinax et al., BioTechniques, 12 (6): 864-869, 1992; and Sawai et al., AJRI, 34: 26-34, 1995; and Better et al., Science, 240: 1041-1043, 1988. Examples of techniques that can be used to produce Fvs and single chain antibodies include those described in US Pat. No. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu et al., PNAS, 90: 7995-7999, 1993; and Skerra et al., Science, 240: 10381040, 1988.

For some uses, which include the in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies that have a variable region derived from a murine monoclonal antibody and a constant region derived from a human immunoglobulin. Methods of producing chimeric antibodies are known in the art. See, for example, Morrison, Science, 229: 1202, 1985; Oi et al., BioTechniques, 4: 214 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. patents No. 5,807,715; 4,816,567; and 4,816,397. Humanized antibodies are antibody molecules of non-human species that bind the desired antigen that have one or more complementarity determining regions (CDRs) of the non-human species and structural regions of a human immunoglobulin molecule. Frequently, residues of the structural region in the human structural regions will be substituted with the corresponding moiety of the CDR donor antibody to preferentially alter, binding to the antigen. These structural region substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and structural region residues to identify residues of the structural region important for antigen binding and sequence comparison to identify unusual remains of the structural region in particular positions. See, for example, Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature, 332: 323, 1988. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR grafting (EP 239,400; PCT Publication WO 91/09967; US Pat. U.S. 5,225,539; 5,530,101 and 5,585,089), surface inactivation or conditioning (EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28 (4/5): 489-498, 1991; Studnicka et al., Protein Engineering, 7 (6): 805-814, 1994; Roguska et al., Proc Natl. Acad. Sci. USA, 91: 969-973, 1994) and chain shuffling (US Pat. No. 5,565,332).

Particularly human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art that include the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See US patents. No. 4,444,887 and 4,716,111; Y

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The modified IgG nucleotide sequence and polynucleotides encoding it can be obtained by any method known in the art, which includes general method of DNA sequencing, such as dideoxy chain termination method (Sanger sequencing), and priming of oligonucleotides in combination with PCR, respectively.

5.2. Identification of mutations within the hinge-Fc region of immunoglobulin molecules

One or more modifications may be made to amino acid residues 251-256, 285-290, 308-314, 385-389 and 428-436 of the constant domain using any technique known to those skilled in the art. The constant domain or fragment thereof having one or more modifications to amino acid residues 251-256, 285290, 308-314, 385-389 and 428-436 can be screened, for example, by a binding assay to identify the domain constant or fragment thereof with high affinity for the FcRn receptor (for example, as described in Section 5.11, below). Those modifications in the hinge-Fc domain or fragments thereof that increase the affinity of the constant domain or fragment thereof for the FcRn receptor can be introduced into antibodies to increase the in vivo half-lives of said antibodies. In addition, those modifications in the constant domain or the fragment thereof that increase the affinity of the constant domain or fragment thereof for FcRn can be fused with bioactive molecules to increase the in vivo half-lives of said bioactive molecules (and, preferably alter (increase or decrease) ) the bioavailability of the molecule, for example, to increase or decrease transport to mucosal surfaces (or other target tissue) (for example, the lungs).

5.2.1. Mutagenesis

Mutagenesis can be performed according to any of the techniques known in the art that include, but are not limited to, synthesizing an oligonucleotide that has one or more modifications within the sequence of the constant domain of an antibody or a fragment thereof (e.g., the domain CH2 or CH3) to be modified. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, in addition to a sufficient number of adjacent nucleotides, to provide a first sequence of sufficient sequence size and complexity. to form a stable duplex on both sides of the deletion junction that is traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. Several such primers that introduce a variety of different mutations in one or more of the positions can be used to generate a library of mutants.

The site-specific mutagenesis technique is well known in the art, as exemplified by various publications (see, for example, Kunkel et al., Methods Enzymol., 154: 367-82, 1987). In general, site-directed mutagenesis is performed by first obtaining a single stranded vector or by fusing the two strands of a double stranded vector that includes within its sequence a DNA sequence encoding the desired peptide. An oligonucleotide primer is prepared that carries the desired mutated sequence, generally synthetically. This primer is then hybridized with the single stranded vector, and subjected to DNA polymerization enzymes such as T7 DNA polymerase, in order to complete the synthesis of the strand that carries the mutation. Thus, a heteroduplex is formed in which one strand encodes the original non-mutated sequence and the second strand carries the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones that include recombinant vectors that carry the mutated sequence arrangement are selected. As will be appreciated, the technique normally employs a phage vector that exists in both a single-stranded and double-stranded form. Typical useful vectors in site-directed mutagenesis include vectors such as phage M13. These phages are readily commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site-directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.

Alternatively, the use of PCR ™ with commercially available thermostable enzymes such as Taq DNA polymerase can be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. See, for example, Tomic et al., Nucleic Acids Res., 18 (6): 1656, 1987, and Upender et al., Biotechniques, 18 (1): 29-30, 32, 1995, for mutagenesis procedures PCR ™ mediated. PCR ™ can also be used using a thermostable ligase, in addition to a thermostable polymerase, to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector (see, for example, Michael, Biotechniques , 16 (3): 410-2, 1994).

Other methods known to those skilled in the art of producing sequence variants of the Fc domain of an antibody or a fragment thereof can be used. For example, recombinant vectors encoding the amino acid sequence of the constant domain of an antibody or a fragment thereof with mutagenic agents, such as hydroxylamine, can be treated to obtain sequence variants.

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techniques well known in the art. Thus, methods of preparing a protein expressing a polynucleotide containing an antibody encoding the nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors that contain antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. The invention thus provides replicable vectors comprising a nucleotide sequence that encodes the constant region of the antibody molecule with one or more modifications to the amino acid residues involved in the interaction with FcRn (see, for example, PCT WO publication 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464). The nucleotide sequence encoding the heavy chain variable region, light chain variable region, both heavy chain and light chain variable regions, an epitope binding fragment of the heavy and / or light chain variable region,

or one or more complementarity determining regions (CDR) of an antibody can be cloned into such a vector for expression.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody that has a high affinity for FcRn and a high half-life in vivo. Thus, the invention includes host cells containing a polynucleotide encoding an antibody, a constant domain that has one or more modifications to amino acid residues 251-256, 285-290, 308-314, 385-389 and / or 428- 436, preferably, operatively linked to a heterologous promoter.

A variety of host-expression vector systems can be used to express the antibody molecules of the invention. Such host-expression systems represent vehicles through which the coding sequences of interest can be produced and subsequently purified, but they also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in if you. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; and mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 and NSO cells) that house recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian virus (for example, the late promoter of adenovirus; the 7.5K promoter of the variolovaccine virus). Preferably, bacterial cells such as Escherichia coli are used, and more preferably, eukaryotic cells, especially for the expression of the complete recombinant antibody molecule, for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the promoter element of the main intermediate early human cytomegalovirus gene, is an effective expression system for antibodies (Foecking et al. , Gene, 45: 101, 1986, and Cockett et al., Bio / Technology, 8: 2, 1990).

In bacterial systems, several expression vectors that depend on the intended use for the antibody molecule that is expressed can be advantageously selected. For example, when a large amount of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruther et al., EMBO, 12: 1791, 1983), in which the antibody coding sequence can be individually bound in the frame vector. with the lacZ coding region so that a fusion protein is produced; and pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13: 3101-3109, 1985 and Van Heeke & Schuster, J. Biol. Chem., 24: 5503-5509, 1989).

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually in non-essential regions (for example, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (for example, the polyhedrin promoter).

In mammalian host cells, several virus-based expression systems can be used to express an antibody molecule of the invention. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may bind to an adenovirus transcription / translation control complex, for example, the late promoter and the tripartite conductive sequence. This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo. Insertion into a non-essential region of the viral genome (eg, E1 or E3 region) will produce a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (for example, see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81: 355-359, 1984). Specific initiation signals may also be required for the

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heavy chain polypeptides as light. In such situations, the light chain must be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, Nature, 322: 52, 1986; and Kohler, Proc. Natl. Acad. Sci. USA, 77: 2 197, 1980). The coding sequences for heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by recombinant expression, it can be purified by any method known in the art for the purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after purification in Protein A, and molecular exclusion chromatography), centrifugation, differential solubility or by any other standard technique for protein purification. In addition, the antibodies of the present invention or fragments thereof can be fused with heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

5.3.1. Antibody conjugates

The present invention encompasses recombinantly fused or chemically conjugated antibodies (including both covalent and non-covalent conjugates) with heterologous polypeptides (i.e., an unrelated polypeptide; or portion thereof, preferably at least 10, at least 20, at least 30, at minus 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins. Fusion does not necessarily have to be direct, but it can occur through connecting sequences. Antibodies fused or conjugated with heterologous polypeptides can also be used in in vitro immunoassays and purification methods using methods known in the art. See, for example, PCT Publication No. WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett., 39: 91-99, 1994; U.S. Patent 5,474,981; Gillies et al., PNAS, 89: 1428-1432, 1992; and Fell et al., J. Immunol., 146: 24462452, 1991.

Antibodies can be fused with marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the label provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of the which are commercially available. As described in Gentz et al., Proc. Natl Acad. Sci. USA, 86: 821-824, 1989, for example, hexa-histidine provides convenient purification of the fusion protein. Other peptide brands useful for purification include, but are not limited to, the hemagglutinin "HA" brand, which corresponds to an epitope derived from the hemagglutinin influenza protein (Wilson et al., Cell, 37: 767 1984) and the "flag" brand (Knappik et al., Biotechniques, 17 (4): 754-761, 1994).

The present invention also encompasses antibodies conjugated with a diagnostic or therapeutic agent or any other molecule for which it is desired to increase the half-life in vivo. Antibodies can be used diagnostically to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical trial procedure to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals and non-radioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated either directly to the antibody or indirectly by an intermediate product (such as, for example, a connector known in the art) using techniques known in the art. See, for example, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, βgalactosidase or acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; An example of a luminescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin and aecuorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99mTc.

An antibody can be conjugated to a therapeutic moiety such as a cytotoxin (for example, a cytostatic agent

or cytocidal), a therapeutic agent or a radioactive element (for example, alpha emitters, gamma emitters, etc.). Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, doxorubicin, daunorubicin, dione dihidroxiantracina, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (for example, mechlorethamine, thioepa, chlorambucil, melphalan (carmustine, carmustine BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (formerly daunomycin) and doxorubicin for example) , dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)), and antimitotic agents (for example, vincristine and vinblastine).

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dideoxynucleotide sequencing (Sanger et al., Proc. Natl. Acad. Sci USA, 74: 5463-5467, 1977) using an ABI3000 genomic analyzer (Applied Biosystems, Foster City, CA).

As a result of immunopurification, two mutants were isolated from the phage library containing mutations in residues 308-314 (fixed H310 and W313), thirteen mutants from the library for residues 251-256 (fixed I253), six mutants from the library for remains 428-436 (H429, E430, A431, L432 and H435 fixed) and nine mutants of the library for remains 385-389 (fixed E388). The mutants isolated from the libraries are listed in Table VIII.

Table VIII

MUTANTS ISOLATED BY IMMUNOPURIFICATION

LIBRARY

MUTANTS *

251-256

Leu Tyr Ile Thr Arg Glu (SEQ ID NO: 90)

Leu Tyr Ile Ser Arg Thr (SEQ ID NO: 91)

Leu Tyr Ile Ser Arg Ser (SEQ ID NO: 92)

Leu Tyr Ile Ser Arg Arg (SEQ ID NO: 93)

Leu Tyr Ile Ser Arg Gln (SEQ ID NO: 94)

Leu Trp Ile Ser Arg Thr (SEQ ID NO: 95)

Leu Tyr Ile Ser Leu Gln (SEQ ID NO: 96)

Leu Phe Ile Ser Arg Asp (SEQ ID NO: 97)

Leu Phe Ile Ser Arg Thr (SEQ ID NO: 98)

Leu Phe Ile Ser Arg Arg (SEQ ID NO: 99)

Leu Phe Ile Thr Gly Ala (SEQ ID NO: 100)

Leu Ser Ile Ser Arg Glu (SEQ ID NO: 101)

Arg Thr Ile Ser Ile Ser (SEQ ID NO: 102)

308-314

Thr Pro His Ser Asp Trp Leu (SEQ ID NO: 103)

Ile Pro His Glu Asp Trp Leu (SEQ ID NO: 104)

385-389

Arg Thr Arg Glu Pro (SEQ ID NO: 105)

Asp Pro Pro Glu Ser (SEQ ID NO: 106)

Ser Asp Pro Glu Pro (SEQ ID NO: 107)

Thr Ser His Glu Asn (SEQ ID NO: 108)

Ser Lys Ser Glu Asn (SEQ ID NO: 109)

His Arg Ser Glu Asn (SEQ ID NO: 110)

Lys Ile Arg Glu Asn (SEQ ID NO: 111)

Gly Ile Thr Glu Ser (SEQ ID NO: 112)

Ser Met Ala Glu Pro (SEQ ID NO: 113)

428-436

Met His Glu Ala Leu Arg Tyr His His (SEQ ID NO: 114)

Met His Glu Ala Leu His Phe His His (SEQ ID NO: 15)

Met His Glu Ala Leu Lys Phe His His (SEQ ID NO: 116)

Met His Glu Ala Leu Ser Tyr His Arg (SEQ ID NO: 117)

Thr His Glu Ala Leu His Tyr His Thr (SEQ ID NO: 118)

Met His Glu Ala Leu His Tyr His Tyr (SEQ ID NO: 119)

* Substitution remains are indicated in bold

10 The sequences underlined in Table VIII correspond to sequences that occurred 10 to 20 times in the final round of immunopurification and the sequences in italics correspond to sequences that occurred 2 to 5 times in the final round of immunopurification. Those sequences that are neither underlined nor in italics occurred once in the final round of immunopurification.

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6.4 Expression and purification of the hinge-Fc region of soluble mutants

The genes encoding mutated hinge-Fc fragments with appropriate restriction enzymes are cleaved and reclining into an expression vector, for example, VβpelBhis (Ward, J. Mol. Biol., 224: 885-890, 1992). 20 vectors can be used that contain any other type of tag sequence, such as c-myc tag, decapeptide tag (Huse et al., Science, 246: 1275-1281, 1989), Flag ™ tags (Immunex). Recombinant clones, such as E. coli, are cultured and induced to express soluble hinge-Fc fragments, which can be isolated from the culture media or cell lysate after osmotic shock, based on the brand used, or by any other method of purification well known to those skilled in the art and characterized by the methods listed at

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6.5 Construction, production and purification of IgG1 variants

Representative Fc mutations were incorporated such as I253A, M252Y / S254T / T256E, M252W, M252Y, 30 M252Y / T256Q, M252F / T256D, V308T / L309P / Q311S, G385D / Q386P / N389S, G385R / P38533 / 38738 / 387K N434F / Y436H and N434F / Y436 in the human IgG1 MEDI-493 (SYNAGIS®) (Johnson et al., 1997, above). The

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Claims (1)

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